Difference between revisions of "Team:OUC-China/Model"

The endoribonuclease Csy4 from CRISPR family is the main role of miniToe system. Csy4 (Cas6f) is a 21.4 kDa protein which recognizes and cleaves a specific 22nt RNA hairpin which consists of an N-terminal ferredoxin-like domain and a C-terminal domain. This later domain constitutes most of the recognition interactions with the RNA. The RNA adopts a stem-loop structure (the specific 22nt RNA hairpin) with five base pairs in A-form helical stem capped by GUAUA loop containing a sheared G11-A15 base pair and a bulged nucleotide U14. In the binding structure of Csy4-RNA complex, the RNA stem-loop straddles the β-hairpin formed by strands β6-7 of Csy4. The Fig.1 and Fig.2 shows two structure of Csy4: with and without hairpin bound.

Based on function of Csy4, we design a new cis-regulatory RNA element named miniToe which can be recognized by Csy4. The whole system works as a translational activator including three modular parts:

1. A cis-repressive RNA (crRNA) to serve as translation suppressor by pairing with RBS as the critical part of miniToe structure.
2. A Csy4 site as a linker between cis-repressive RNA and RBS, which can be specifically cleaved upon Csy4 function.
3. A CRISPR endoribonuclease Csy4.

Fig.2-1 is the secondary structure of miniToe.


The Fig.4 and Fig.5 show that the two complex of miniToe structure: with and without specific site of hairpin cleaved, which is called the precursor complex and precursor complex respectively.

All the reaction happened in our first system, miniToe, can be described chronologically by following five main steps in Fig.6:

(1)The miniToe structure is produced and accumulated.
(2)The Csy4 is produced with IPTG induced.
(3)The Csy4 binds to the miniToe structure and form the Csy4-miniToe complex
(4)The Csy4 cleave the special site and divide the miniToe structure into two parts: the Csy4-crRNA complex and the mRNA of sfGFP.
(5)The sfGFP is produced.

From the description above, we can get four key problems in our system to make sure that our system can work successfully:

(1)Does the Csy4 dock correctly with the miniToe structure (hairpin)?
(2)How about the ability of binding between the Csy4 and miniToe structure (hairpin)?
(3)How about the ability of cleavage between the Csy4 and miniToe structure (hairpin)?
(4)Does cis-repressive RNA release from the RBS?

According to the work process we build an ODEs model and simulate our miniToe system for 30h, the result can be seen in the Fig.7.

We compare the experimental data to the simulation, find it fit perfectly in Fig.7
  To see more details

The main work our molecular dynamics is to give an explanation for the experimental data at atom level and four key points we discuss before: the experiment have proved that the miniToe structure is working well which means the four key points is work well too. We will present you the result of molecular dynamics following the four key points.

For the first key point, we have the interaction matrix to describe the molecular docking, and the heatmap of matrix can be seen in Fig.9.


For the second problem, we calculated the free binding energy of Csy4/RNA complex. The result of binding free energy for wild-type Csy4 is $△G_{binding}=-59154.9251\stackrel{}{}kj/\text{mol}$ .

For the third key point, we check the distance of Ser151(OG)-G20(N2’), which is a key interaction in the active site of Csy4 to describe the ability of cleavage. The distance curve of Ser151(OG)-G20(N2’) for wild-type Csy4 can be seem in Fig.10.




For the last key points, we use the RMSD of product to describe the release of crRNA. The result can be seen in the Fig.11.
The RMSD is unstable which give an explain to experiment that crRNA is release from RBS.

  To see more details

When we make the sensitivity analysis for our ODEs model, we find that while the cleavage rate of Csy4 will influence the expression in GFP, which indicated if we change the wild-type Csy4 to some mutation then we can achieve the different expression of GFP. The Fig.12 is showing that what will happen in the GFP expression curve if we change the cleavage rate of Csy4. It can achieve the goal of different regulate level.

The wet lab members give us four important sites, Gln104, Tyr176, Phe155, His29, which play important roles in binding and cleavage in protein Csy4 which can be seen in Fig.13. Considering 20 kinds of amino acids, we have 80 mutants to explore and choose if we only have one site mutated.


In Q3, we point out four key points which will directly influence the work of our miniToe system. And in Q5, according to the

Now we are going to construct an logic line to show you how to use the three main information above to designing the Csy4 mutnats:

What we know and proved by the experiment is that the wild-type Csy4 with the miniToe system is working well, which means that all the important key points we discussion did not exist in the wild-type Csy4. The wild-type Csy4 can dock correctly with the miniToe structure and the Csy4 have a good ability to bind and cleave the miniToe structure, finally the crRNA release from the RBS. So we choose the wild-type Csy4 as a standard, and all the Csy4 mutant can check the four key points by comparing to wild-type Csy4.

Now for the four key points in Q3 we have something in mathetical forms to describe it in Q5. The most important thing is transform to the problem that how to make an compasion between mutant and